Compositions and methods relating to reduction of symptoms of autism

ABSTRACT

Methods and compositions that can reduce the symptoms of autism in a human patient comprising administering a physiologically effective amount of one or both of a purified casomorphin inhibitor selected from the group consisting of a casomorphinase and a casomorphin ligand, and a physiologically effective amount of a purified gluteomorphin inhibitor selected from the group consisting of a gluteomorphinase and a gluteomorphin ligand, to a human patient in sufficient quantities to reduce the effects of the autism. In some embodiments, the compositions and methods further comprise a physiologically effective amount of an enkephalin inhibitor, preferably an enkephalinase, and a physiologically effective amount of an endorphin inhibitor, preferably an endorphinase.

RELATED APPLICATIONS

This application is a continuation of application of U.S. patentapplication Ser. No. 10/237,160, filed Sep. 5, 2002 now U.S. Pat. No.6,808,708, which is a continuation application of U.S. patentapplication Ser. No. 09/411,605, filed Oct. 1, 1999, now U.S. Pat. No.6,447,772, the contents of which are incorporated by reference in theirentirety.

FIELD OF THE INVENTION

The field of the present invention is the reduction of symptoms ofautism

BACKGROUND OF THE INVENTION

Autism is a relatively rare syndrome of early childhood that affectsbetween three and eight of every 10,000 school-aged children. Autism isa serious disease that seriously impairs the functioning andlife-enjoyment of its victims. The disease can include languagedisorders with impaired understanding, echolalia, pronominal reversal(such as using “you” instead of “I” or “me” when referring to one'sself), rituals and compulsive phenomena, and uneven intellectualdevelopment with mental retardation. Autism is about two to five timesmore common in boys than in girls. The cause of autism is unknown, butthere are, at the least, some important genetic factors, as indicated bythe fact the concordance rate is significantly greater in monozygotictwins than dizygotic twins. Merck Manual, 17^(th) edition, section 19,chapter 274 (1999); Autism Review, Lowell Ackerman,http://www.parentzone.com/autism/review.htm (1997). Other factors mayinclude rubella, problems during pregnancy, labor and delivery,cytomegalic inclusion disease, phenylketonuria, and fragile X syndrome.Autistic children are also at increased risk of developing seizuredisorders, especially during their teen years.

Analysis of the urine of autistic children found hyperpeptiduria in thechildren, which means that the analysis found a significantly increasedpresence of peptides (short chains of amino acids) in the urine ofchildren. Reichelt et al., J. Applied Nutr., 42(1):1-11 (1990); Reicheltet al., Brain Dysfunct., 4:308-319 (1991); Reichelt et al., Dev. BrainDysfunct,. 7:71-85 (1994). Reichelt et al. (1994) hypothesized that thepeptiduria was caused by insufficient breakdown of peptide fragmentsfrom dietary milk protein (i.e., casein) and wheat protein (i.e.,gluten), resulting in the uptake of the peptides by the body. This isproblematic because the peptides from casein and gluten, casomorphin andgluteomorphin, respectively, have opioid qualities, which means thatthey mimic opiates in the body (indeed, that is indicated by the“-morphin” suffix of the names, which shows their functional similarityto morphine, a strong opiate; casomorphin is also known ascaseomorphine). For example, one molecule of gluten contains 15 opioidsequences, which can be released by the action of trypsin, chymotrypsin,and secretin. Id. Reichelt et al. also hypothesized that the release ofthe opioid-like casomorphin (from casein) and gluteomorphin (fromgluten) were caused by a defect of peptidases in the patient. Reicheltet al. (1991) at 308.

Accordingly, Reichelt et al. proposed a strict gluten-free andcasein-free diet (i.e., strictly wheat-free and dairy-free). Reichelt etal. (1990) found that such a diet ultimately resulted in increasedsocial contact, decreased stereotypy, an end to self-mutilation likehead banging, and a decrease in dreamy state periods. Also, alimentaryproblems generally improved. Reichelt et al. (1990) at 5; accordReichelt et al. (1991); Reichelt et al. (1994).

Ackerman (1997) hypothesized that the addition of papain, bromelain, andchymotrypsin to the diet of the patient might be beneficial. However,Ackerman never reported the actual use of any such enzymes, and hisproposed combination would not be expected to work because chymotrypsin,according to Reichelt et al. (1994) at 79, is one of the digestiveenzymes believed to release the detrimental opioid sequences (as well astrypsin and the hormone secretin). Papain and bromelain are, likewise,broad-spectrum digestive enzymes that would also have a reasonably highchance of actually increasing the amount of opioids, i.e., casomorphinsor gluteomorphins, instead of reducing them (absent the additional useof an agent to specifically inhibit the casomorphin or gluteomorphin, asdiscussed further herein).

As noted above, Reichelt et al. found that a strict gluten-free andcasein-free diet had beneficial effects for the patient. However, asdescribed by Seroussi, Frequently Asked Questions About DietaryIntervention For The Treatment Of Autism And Other DevelopmentalDisabilities, http://www.enabling.org/ia/celiac/aut/autgfffaq.html(1999), achieving and maintaining such a diet can be very difficult: “Becareful. Removing all dairy means ALL milk, butter, cheese, creamcheese, sour cream, etc. It also includes product ingredients such as‘casein’ and ‘whey,’ or words even containing the word ‘casein.’ Readlabels—items like bread and tuna fish often contain milk products. Evensoy cheese usually contains caseinate.” Failure to adhere to the strictdiet can be seriously detrimental: “What you need to understand is thatfor certain children, these [dairy and wheat] foods are toxic to theirbrains . . . . You would never knowingly feed your child poison, but ifhe fits into this category, that is exactly what you could be doing.”Id.; see Reichelt et al. (1991) (abstract; those patients that remainedon the diet had further improvement, while those that abandoned the dietshowed regression).

Thus, there has gone unmet a need for improved methods of treatingpatients with autism who exhibit the effects of exorphins such asgluteomorphin and casomorphin without requiring the patient to adhere todifficult dietary restrictions. Similarly, there has gone unmet a needto protect autistic patients from inadvertent exposure to gluten andcasein, typically in the form of dairy products and wheat products. Thepresent invention provides these and other advantages.

SUMMARY OF THE INVENTION

The present invention provides methods and compositions that can reducethe symptoms of autism in a human patient. Briefly, the methods andcompositions comprise administering a physiologically effective amountof one or both of a purified casomorphin inhibitor selected from thegroup consisting of a casomorphinase and a casomorphin ligand, and aphysiologically effective amount of a purified gluteomorphin inhibitorselected from the group consisting of a gluteomorphinase and agluteomorphin ligand to a human patient in sufficient quantities toreduce the effects of the autism. When administered to human patientssuffering from autism, without restriction on the normal diet of thepatients, the compositions and methods reduced one or more symptoms ofautism, such as increased eye contact, better enunciation and use ofpronouns, less fatigue, singing a song for the first time with themelody and words together and the entire song understandable, playingwith age appropriate friends for the first time, fewer tantrums, bettersleep patterns, improved politeness and coordination, being more loving,acknowledging another individual's emotion, increased voice and wordassociation, and, in one case, noticing that a calendar needed changing.In addition, the present invention provides compositions and methodsthat inhibit gluteomorphin and casomorphin, and other exorphins, fromsources other than casein and gluten, which assists in the treatment ofautism for persons that are already wheat-free and dairy-free, yet arestill ingesting, or otherwise taking in, exorphins from other sources.

Thus, in one aspect the present invention provides compositions able toreduce the symptoms of autism in a human patient, comprising aphysiologically effective amount of a purified casomorphin inhibitorselected from the group consisting of a casomorphinase and a casomorphinligand, a physiologically effective amount of a purified gluteomorphininhibitor selected from the group consisting of a gluteomorphinase and agluteomorphin ligand, and at least one of the group consisting of aphysiologically acceptable carrier, adjuvant, excipient, buffer anddiluent. In a preferred embodiment, the casomorphinase is a prolineprotease, further preferably a protease comprising the dipeptidaseactivity of dipeptidyl peptidase IV. (The present invention comprisesmultiple aspects, features and embodiments; such multiple aspects,features and embodiments can be combined and permuted in any desiredmanner.)

In another embodiment, the compositions comprise the casomorphin ligandor the gluteomorphin ligand, or the gluteomorphinase is selected fromthe group consisting of a tyrosinase and a phenylalaninase. Thecomposition can be suitable for oral administration, and in onepreferred embodiment the casomorphin inhibitor and the gluteomorphininhibitor are not enterically coated, although they can also beenterically coated if desired. Also preferably, the casomorphininhibitor comprises a microbial proline peptidase and the gluteomorphininhibitor comprises a microbial gluteomorphinase selected from the groupconsisting of a tyrosinase and a phenylalaninase. At least one of thegroup consisting of the physiologically acceptable carrier, adjuvant,excipient, buffer and diluent comprises a carbohydrate, for examplemaltodextrin, L-lysine and lactase.

In other embodiments, composition further comprises a physiologicallyeffective amount of an enkephalin inhibitor, preferably an enkephalinaseand a physiologically effective amount of an endorphin inhibitor,preferably an endorphinase.

In another aspect, the present invention provides methods of reducingthe symptoms of autism in a human patient, comprising administering tothe patient a composition comprising a physiologically effective amountof a purified casomorphin inhibitor selected from the group consistingof a casomorphinase and a casomorphin ligand, a physiologicallyeffective amount of a purified gluteomorphin inhibitor selected from thegroup consisting of a gluteomorphinase and a gluteomorphin ligand, andat least one of the group consisting of a physiologically acceptablecarrier, adjuvant, excipient, buffer and diluent.

In a further aspect, the present invention provides methods ofmanufacturing a medicament able to reduce the symptoms of autism in ahuman patient, comprising combining a physiologically effective amountof a purified casomorphin inhibitor selected from the group consistingof a casomorphinase and a casomorphin ligand, a physiologicallyeffective amount of a purified gluteomorphin inhibitor selected from thegroup consisting of a gluteomorphinase and a gluteomorphin ligand, andat least one of the group consisting of a physiologically acceptablecarrier, adjuvant, excipient, buffer and diluent

In yet another aspect, the present invention provides compositions ableto reduce the symptoms of autism in a human patient, comprising aphysiologically effective amount of a purified casomorphin inhibitor, aphysiologically effective amount of a purified gluteomorphin inhibitor,and at least one of the group consisting of a physiologically acceptablecarrier, adjuvant, excipient, buffer and diluent, wherein thecasomorphin inhibitor comprises a proline protease grown by a processselected from the group consisting of tray fermentation and deep-tankfermentation and purified by a process comprising alcohol precipitationand filtration, and wherein the gluteomorphin inhibitor comprises atyrosinase purified by the process of grown by a process selected fromthe group consisting of tray fermentation and deep-tank fermentation andpurified by a process comprising alcohol precipitation and filtration.

In still a further aspect, the present invention provides methods ofreducing the symptoms of autism in a human patient, comprisingadministering to the patient a composition comprising a physiologicallyeffective amount of a purified casomorphin inhibitor, a physiologicallyeffective amount of a purified gluteomorphin inhibitor, and at least oneof the group consisting of a physiologically acceptable carrier,adjuvant, excipient, buffer and diluent, wherein the casomorphininhibitor comprises a proline protease purified by the process of grownby a process selected from the group consisting of tray fermentation anddeep-tank fermentation and purified by a process comprising alcoholprecipitation and filtration, and wherein the gluteomorphin inhibitorcomprises a tyrosinase or a phenylalaninase purified by the process ofgrown by a process selected from the group consisting of trayfermentation and deep-tank fermentation and purified by a processcomprising alcohol precipitation and filtration.

In yet still another aspect, the present invention provides compositionsable to reduce the symptoms of autism in a human patient, comprising ameans to inhibit casomorphin, a means to inhibit gluteomorphin, and atleast one of the group consisting of a physiologically acceptablecarrier, adjuvant, excipient, buffer and diluent.

In another aspect, the present invention provides methods of reducingthe symptoms of autism in a human patient, comprising a step ofadministering to the patient a composition comprising a means to inhibitcasomorphin, a means to inhibit gluteomorphin, and at least one of thegroup consisting of a physiologically acceptable carrier, adjuvant,excipient, buffer and diluent.

In still a further aspect, the present invention provides foods and foodsupplements comprising compositions as described herein.

In yet another aspect, the present invention provides kits comprising avessel containing compositions as described herein and instructionsdirecting the use of the composition to reduce autistic symptoms in ahuman patient of an exorphin selected from the group consisting of agluteomorphin and a caseomorphin. In another embodiment, the presentinvention provides vessels containing such compositions and a labelattached to the vessel comprising instructions directing the use of thecomposition to reduce the symptoms. These and other aspects, featuresand embodiments of the present invention will become evident uponreference to remainder of this application, including the followingDetailed Description and attached drawings. In addition, variousreferences are set forth herein that describe in more detail certaincompositions, apparatus and/or methods; all such references areincorporated herein by reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a method for the preparation of an exorphinase.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods and compositions able to reducethe symptoms of autism in a patient, including a human patient. Briefly,the compositions and methods comprise administering a casomorphininhibitor and/or gluteomorphin inhibitor to a human patient insufficient quantities to reduce the effects of the autistic disease. Aninitial trial wherein casomorphin and gluteomorphin inhibitors wereadministered to human patients, without restriction on the normal dietof the patients, provided a significant number of the patients with asignificant reduction of one or more symptoms, such as increased eyecontact, better enunciation and use of pronouns, less fatigue, singing asong for the first time with the melody and words together and theentire song understandable, playing with age appropriate friends for thefirst time, fewer tantrums, better sleep patterns, improved politenessand coordination, being more loving, acknowledging another individual'semotion, increased voice and word association, and, in one case,noticing that a calendar needed changing.

The following paragraphs provide definitions of some of the terms usedherein. All such terms, including those specifically described below inthis section, are used in accordance with their ordinary meanings unlessthe context or definition indicates otherwise. Also unless indicatedotherwise, except within the claims, the use of “or” includes “and” andvice-versa; similarly, non-limiting terms are not to be construed aslimiting unless expressly stated (for example, “including” means“including without limitation”).

“Composition” indicates a combination of multiple substances into anaggregate mixture.

A “purified” component of a composition, such as an exorphin inhibitor,indicates that the substance is more pure than when it occurs in itsnatural state, but it may potentially contain other enzymes and otheractive material. For example, a purified casomorphin inhibitor obtainedfrom a cellular culture is more pure than when it was in existence inits originating cell, but may still contain other substances such as agluteomorphin inhibitor, lactases, and other cellular material. “Highlypurified” indicates that no other substances having significantbiological activity, such as enzymatic activity, are present in thehighly purified material (the highly purified material may have thepresence of detectable but non-physiologically effective amounts ofother activities).

A “physiologically effective amount” of an active substance such as anexorphin inhibitor indicates an adequate amount of the active substancesto have a significant, externally observable effect on the patient.Thus, such a physiologically effective amount affects one or more of thecharacteristics in the patient without the need for special equipment todetermine the effect. For example, a physiologically effective amount ofan exorphin inhibitor has a significant, externally observable reductionof effect on the opioid-like bioactivity of the exorphin, and thusreduces one or more of the symptoms of autism in a human patient withoutthe need for special equipment to determine the effect. Accordingly, onecan determine whether an adequate amount of the active substance hasbeen administered by watching the patient and observing whether changeshave occurred in the patient due to the active substance.

“Protein” indicates an extremely complex combination of amino acids in alengthy chain(s). Proteins are an essential constituent of living cells.“Proteases” are enzymes that act upon proteins to alter their structureor composition, for example by reducing them, oxidizing them or cleavingthem into smaller chains of amino acids, typically without the enzymesthemselves being consumed in the reaction.

“Peptide” indicates a small chain of amino acids, often derived from thebreakdown of proteins. Peptides are typically comprised of two or moreamino acids, but typically less than 10 or 20 amino acids. A“peptidase,” like a protease, is an enzyme that acts on the peptide toalter the structure or composition of the peptide. An exopeptidase (likean exoprotease) acts on amino acids at the end of an amino acid chain,typically by cleaving them from the remainder of the chain. Anendopeptidase (like an endoprotease) acts on amino acids within themiddle of an amino acid chain. Thus, an exopeptidase can shorten anamino acid chain, while an endopeptidase can give multiple chains ofamino acids from the initial substrate. A dipeptidase is a type ofexopeptidase that cleaves at the penultimate position from the terminusof the peptide chain, thereby cleaving the peptide such that two aminoacids are cleaved at one time from the remainder of the peptide.

“Exorphins” are external substances that have opiate-like (narcotic)activity in the body, thereby acting like the body's own narcotics, theendorphins. The two primary examples of exorphins relevant to thepresent application are casomorphins and gluteomorphins. By providingexorphin inhibitors, the present invention inhibits the opioidbioactivity of exorphins, by action of an exorphinase, which alters thestructure or composition of the exorphins, for example by oxidation,reduction or cleavage, or by action of an exorphin ligand, which bindsto the exorphin.

“Casomorphin” is an exorphin derived from casein via the activity ofproteases, possibly including chymotrypsin and trypsin, as well asindirectly by the action of gastric hormones such as secretin.Casomorphins typically comprise the amino acid sequence Tyr-Pro-Phe-Pro(SEQ ID NO:1).

A “casomorphin inhibitor” is a substance that inhibits the opioid-typebioactivity of casomorphin. The present invention is directed to twotypes of casomorphin inhibitors, casomorphinases and casomorphinligands. A “casomorphinase” is an enzyme that inhibits the opioid-typebioactivity of casomorphin by altering the structure or composition ofthe casomorphin. For example, the casomorphinase may affect casomorphinby oxidation (the casomorphinase is an oxidase), reduction (a reductase)or cleavage (by action of a peptidase). A casomorphin ligand is amolecule that binds to the casomorphin such that the casomorphinsubstantially loses its opioid-type bioactivity.

“Gluteomorphin” is an exorphin derived from gluten via the activity ofproteases, possibly including chymotrypsin and trypsin, as well asindirectly by the action of gastric hormones such as secretin.Gluteomorphins typically comprises an amino acid sequence ofGly-Tyr-Tyr-Pro-Thr (SEQ ID NO:2), Gly-Phe-Phe-Pro (SEQ ID NO:3),Phe-Gly-Gly-Tyr-Leu (SEQ ID NO:4), or Phe-Gly-Gly-Tyr (SEQ ID NO:5).According to Reichelt et al. (1991) one molecule of gluten containsabout 15 different gluteomorphin, i.e., opioid, sequences.

A “gluteomorphin inhibitor” is a substance that inhibits the opioid-typebioactivity of gluteomorphin. The present invention is directed to twotypes of gluteomorphin inhibitors, gluteomorphinases and gluteomorphinligands. A “gluteomorphinase” is an enzyme that inhibits the opioid-typebioactivity of gluteomorphin by altering the structure or composition ofthe gluteomorphin. For example, the gluteomorphinase may affectgluteomorphin by oxidation (the gluteomorphinase is an oxidase),reduction (a reductase) or cleavage (by action of a peptidase). Agluteomorphin ligand is a molecule that binds to the gluteomorphin suchthat the gluteomorphin substantially loses its opioid-type bioactivity.

A “proline protease” is a protease that cleaves a protein or a peptideon the basis of the presence of a proline amino acid in the sequence ofthe protein or peptide. “Dipeptidyl peptidase IV” (“DPP IV”) is aDipeptidyl peptidase that cleaves peptides comprising a proline at thepenultimate position at the amino-terminus of the peptide. Handbook ofProteolytic Enzymes,” CLAN SC-S9, § 128, p. 378-382 (Academic Press,Barrett, et al., eds., 1998). Similarly, a “tyrosinase” is a proteasethat cleaves, oxidizes and/or reduces a protein on the basis of atyrosine in the protein. “Phenylalaninase” is an example of anotherexomorphinase, which cleaves, oxidizes and/or reduces a protein on thebasis of a phenylalanine in the protein.

A “microbe” means microscopic organisms, including organisms such asbacteria and fungi.

“Enkephalin” indicates a naturally occurring substance in the brain,typically either of two penta-peptides, with opiate and analgesicactivity and a marked affinity for opiate receptors. An enkephalininhibitor is a substance that inhibits the bioactivity of an enkephalinsuch that the inhibition has an observable effect on a patient whoseenkephalins have been so inhibited. An “enkephalinase” is an enzyme thatoxidizes, reduces, cleaves or otherwise alters the structure orcomponents of enkephalin such that it substantially loses its opiateactivity. An “enkephalin ligand” is a substance that binds to theenkephalin such that the enkephalin substantially loses its opiate-typebioactivity.

An “endorphin” is any of a group of proteins with potent narcotic oranalgesic properties that occur naturally in the brain. An endorphininhibitor is a substance that inhibits the bioactivity of an endorphinsuch that the inhibition has an observable effect on a patient whoseendorphins have been so inhibited. An “endorphinase” is an enzyme thatoxidizes, reduces, cleaves or otherwise alters the structure and/orcomponents of endorphin such that it substantially loses its opiateactivity. An “endorphin ligand” is a substance that binds to theendorphin such that the endorphin substantially loses its opiate-typebioactivity.

The terms set forth in this application are not to be interpreted in theclaims as indicating a “means plus function” relationship unless theword “means” is specifically recited in a claim, and are to beinterpreted in the claims as indicating a “means plus function”relationship where the word “means” is specifically recited in a claim.Similarly, the terms set forth in this application are not to beinterpreted in method or process claims as indicating a “step plusfunction” relationship unless the word “step” is specifically recited inthe claims, and are to be interpreted in the claims as indicating a“step plus function” relationship where the word “step” is specificallyrecited in a claim.

Other terms and phrases in this application are defined in accordancewith the above definitions, and in other portions of this application.

Turning to a more detailed discussion of the invention, in a firstaspect the present invention provides compositions that are able toreduce the symptoms of autism in a human patient. For example, thecompositions are able to reduce one or more symptoms, such as increasedeye contact, better enunciation and use of pronouns, less fatigue, fewertantrums, better sleep patterns, improved politeness and coordination,and increased voice and word association. In other words, thecompositions are able to effect an adequate reduction of one or more ofthe observable characteristics of autism by an amount that is observableto a human observer, such as a parent, physician or caretaker, withoutthe use of special devices such as microscopes or chemical analyticaldevices. The compositions reduce such symptoms by providing aphysiologically effective amount of a purified casomorphin inhibitorselected from the group consisting of a casomorphinase and a casomorphinligand and a physiologically effective amount of a purifiedgluteomorphin inhibitor selected from the group consisting of agluteomorphinase and gluteomorphin ligand, and also at least one of thegroup consisting of a physiologically acceptable carrier, adjuvant,excipient, buffer and diluent, which terms are used in their ordinarysense to indicate substances that assist in the packaging, delivery,absorption, or, in the case of an adjuvant, enhancing the physiologicaleffect of one or both of the casomorphin inhibitor and the gluteomorphininhibitor.

The physiologically acceptable carriers, adjuvants, excipients, buffersand diluents are preferably nontoxic to recipients at the dosages andconcentrations employed. Representative samples include water, isotonicsaline solutions that are preferably buffered at physiological pH (suchas phosphate-buffered saline or Tris-buffered saline), mannitol,dextrose, glycerol, and ethanol, as well as selected polypeptides orproteins such as human serum albumin, maltodextrin, L-lysine, lactaseand other carbohydratases, lipase and non-specific proteases such aspapain. The carrier, adjuvant, excipient, buffer, or diluent may becombined with the exorphin inhibitors to provide compositions either asliquid solutions or, preferably, in solid form. For example, when thecompositions are to be administered orally, the compositions may beproduced in any of powder, tablet or capsule form.

The compositions of the present invention are preferably administeredorally, but may also be administered via other direct routes, such asrectal or, in the case of pharmaceutically designed compositions, viatranscutaneous methods such as intraarterial, intramuscular,intraperitoneal, subcutaneous, intraocular, and intravenous. Otherroutes such as buccal/sublingual, nasal, topical (such as transdermaland hypothalamic), vaginal and pulmonary may also be used, if desired.The compositions are typically administered to human beings, but mayalso be administered to animals, preferably mammals, displaying symptomssimilar to autism.

Turning to some of the other components of the compositions, theexorphin inhibitors, i.e., the casomorphin inhibitors and thegluteomorphin inhibitors, are present in the composition in adequateamounts to reduce one or more symptoms of autism in at least aboutone-fourth of the human patients to whom the composition isadministered. Preferably, the composition reduces the symptoms of two ormore symptoms of autism (or even up to 10 or more, or all), and furtherpreferably reduces the symptoms in one-half or more of the humanpatients to which it is administered.

In a preferred embodiment, the casomorphin inhibitor is acasomorphinase, further preferably a proline protease, which means aprotease that acts, typically by cleavage, on the substrate casomorphinbased upon the presence of a proline amino acid residue within thepeptide. Further preferably, the proline protease is a peptidasecomprising a dipeptidase activity similar to that of DPP IV, which meansthat the protease cleaves at the penultimate position of a peptide whena proline is located at such penultimate position. Still furtherpreferably, the proline protease is DPP IV.

In another embodiment, the gluteomorphin inhibitor is agluteomorphinase, preferably a tyrosinase, which means an enzyme thatoxidizes, reduces or cleaves the substrate gluteomorphin due to thepresence of tyrosine residue(s) in the gluteomorphin. Rosei et al.,Recenti Prog. Med., ADH (3):134-139 (1997); Rosei et al, Biochem. Int.,19(6):1183-1193 (1989); Rosei et al., Biochem. Biophys. Acta,1199(2):123-129 (1994); Larsimont et al., Biochem. Biophys. Acta,1222(1):95-100 (1994). Further preferably, the tyrosinase comprises anoxidizing activity of the tyrosine when in the presence of L-dopa(L-dihydroxyphenylalanine)In a further embodiment, the gluteomorphinaseis preferably a phenylalaninase, which means an enzyme that oxidizes,reduces or cleaves the substrate gluteomorphin due to the presence ofphenylalanine residue(s) in the gluteomorphin.

As noted above, the compositions of the present invention can beadministered orally to the patient. In a preferred embodiment, thecasomorphin inhibitors and the gluteomorphin inhibitors of the presentinvention are not enterically coated, which means that the inhibitorshave not been treated with any specific substances to assist the passageof the inhibitors through the rigors of the stomach to the intestines.Thus, in this embodiment, it is preferred to use exorphin inhibitorsable to withstand such caustic stomach conditions. One example ofsuitable exorphin inhibitors are those derived from microbial sourcessuch as bacteria and fungi. Preferred bacterial sources include membersof the Bacillus, including Bacillus amyloliquifaciens, Bacilluscoagulans, Bacillus lichenformis, Bacillus macerans and Bacillussubtilis. Preferred fungi include species of the genus Aspergillus, forexample Aspergillus oryzae, Aspergillus niger, Aspergillus awamori,Aspergillus flavus, Aspergillus japonicus, Aspergillus saitoi,Aspergillus sojae, and Aspergillus usamii shirousami.

In preferred embodiments, one or more, or all, of components of thecomposition are produced from a microbe such as the microbes discussedabove or a recombinant microbe that has been produced by recombinanttechnology, or other methods of introducing foreign genes into a desiredspecies or cell line. Thus, the recombinant microbe produces a desiredagent for use in the compositions and methods of the present invention,such as a desired exomorphin inhibitor, enkephalinase or endorphinase.Preferred examples of such recombinant cell lines include E. coli,Sacchromyces, Candida, and desired mammalian or other eukaryotic celllines. The microbes can be grown, for example, by the processes of solidstate fermentation or deep-tank fermentation. In solid statefermentation, the microbes are typically grown on trays or other solidsubstrates, while in deep-tank fermentation the microbes re raised inlarge tanks using cultures submerged in a solution of oxygenated media.

In one embodiment, such fermentation comprises growing theenzyme-producing organism on a suitable energy-providing substrate suchas koji (wheat or rice bran), which substrate has preferably beensterilized to eliminate unwanted organisms from the fermentationprocess. Thus, the sterile koji or other energy-providing material isinoculated with the desired strain of microbe that will produce thedesired enzymes. Fermentation proceeds under controlled temperature andhumidity conditions from about a few days to a week. At the conclusionof the fermentation, the enzymes are solubilized into an aqueous phaseand substrate is removed by conventional filtration. If desired,microfiltration and/or ultrafiltration steps may also be used toconcentrate the aqueous enzyme prior to precipitation. Where it isdesired to produce powdered enzymes, soluble enzymes can be precipitatedwithin an appropriate precipitating agent, for example an alcohol,preferably a non-toxic alcohol such as ethanol, then washed and dried.Pursuant to the precipitation step, the resulting powder product can beconsidered to be microbially very clean, which indicates that it has avery low level of microbes when compared to other products such aspasteurized (fluid) milk. In addition, the composition preferably isfree of toxins, particularly mycotoxins in the case of fungi, which canbe assayed using tests well known to those of ordinary skill in the artin view of the present disclosure. In addition, alternative methods ofmaking and purifying desired enzymes and other exorphin inhibitors, fromboth microbial and non-microbial sources, and including alternativesolid state fermentation processes, will be readily apparent to a personof ordinary skill in the art in view of the present specification. Forexample, the pH, temperature, buffers, sugars, minerals, and otherparameters of the growth conditions can be adjusted as desired by aperson having ordinary skill in the art in view of the presentspecification to enhance growth for particular desired substances,organisms or cell lines.

In additional embodiments, the compositions can further comprise one orboth of an enkephalin inhibitor and an endorphin inhibitor. Suchinhibitors function in much the same way as the exorphin inhibitors, andtherefore the discussion herein relating to such inhibitors applies tothe enkephalin and endorphin inhibitors as well.

In another aspect, the present invention provides compositionscomprising microbially-derived casomorphinases in combination with aphysiologically acceptable carrier, adjuvant, excipient, buffer ordiluent. As noted above, in preferred embodiments the compositionadditionally comprises one or more of a gluteomorphin inhibitor, anenkephalin inhibitor and an endorphin inhibitor Preferably, theinhibitors are not enterically coated.

In a preferred embodiment, the compositions are provided to the patientas either a food or a food supplement. For example, when provided as afood the compositions of the present invention are combined withmaterial primarily made up of protein, carbohydrate and/or fat that isused in the body, preferably a human body, to sustain growth, repair,vital processes, and to furnish energy. When provided as a foodsupplement, the compositions comprise selected substances such that theycan be eaten at or about the same time as a food. The food supplementsare generally eaten within about one hour before or after the food iseaten, typically within about one-half hour before or after the food iseaten, preferably within about 15 minutes of when the food is eaten, andfurther preferably within one to five minutes of the time the food iseaten. The food supplement can also be eaten at the same time as, oreven with the food.

In another aspect, the present invention provides methods for reducingthe symptoms of autism in a patient, preferably a human patient. In oneembodiment, such methods comprise administering compositions asdescribed elsewhere herein to a patient in a physiologically effectiveamount to reduce one or more symptoms of autism, as described elsewhereherein.

In a preferred embodiment, as noted above, the methods compriseadministering the compositions at or about the time that foodcontaining, or potentially containing, casein or gluten are eaten by apatient, so that the exorphin-inhibitory activities of the compositionwill coincide with the presence of the food in the gut, preferablybeginning in the stomach and continuing on into the intestines.

In an additional aspect, the present invention provides methods ofmanufacturing a medicament able to reduce the symptoms of autism whereinthe medicament comprises the compositions as described elsewhere herein.

In an additional aspect, the present invention provides vesselscomprising compositions as described herein and a label attached to thevessel wherein the label comprises instructions or directions advising apatient to use the composition to reduce the symptoms of autism. Thevessel can be any appropriate container including a can, a vial, a boxor any other appropriate vessel, for example such as a hypodermic needlefor the intravenous administration of the composition. A label can beattached to the vessel via any suitable approach, including, forexample, glue or a string. In a similar aspect, the present inventionalso provides kits comprising a vessel, as described above, andinstructions. In the kit, the instructions need not be physicallyattached to the vessel.

EXAMPLES Example 1 Production of Exorphinase from a Fungus

As depicted in FIG. 1, an exorphinase was produced by first growing adesired microbe in a tube to provide a tube culture which was thenexpanded to a flask culture in a flask 6. The culture was then furtherexpanded in a seed tank 8 and then introduced into a rotating cooker 10containing wheat bran and water. The inoculate was then grown from twoto ten days until a desired level of growth was attained, and then theinoculated wheat bran 12 was removed from the rotating cooker 10 andtransferred to a cultivation chamber 14, which cultivation chamber wasprovided with water, steam and filtered air as desired.

The resulting cultured bran was then transmitted through a crusher 16and then placed into an extractor 18 where it was extracted with water.The extract was placed into a first precipitation tank 20 comprisingdiatomaceous earth and ethanol, and the resulting solution was thentransmitted through a filter press 22 where the resulting cake wasdiscarded and the filtrate was transmitted on to a bacteriologicalfilter 24. The filtrate from the bacteriological filter was thentransmitted into a second precipitation tank 26 containing ethanol toprovide a slurry. The slurry from the second precipitation tank 26 wasthen placed into a dehydration tank 28, also containing ethanol, and theresulting slurry was then centrifuged in a centrifuge 30. The resultingcake from the centrifuge was transferred to a vacuum dryer 32 where itwas dried and then placed into a sifter 34. The material that sifted outwas placed into a blender 38, while material too large to sift was runthrough a pulverizer 36 and then placed into the blender 38. In theblender, a desired diluent was added to provide a finished productcontained in vessel 40.

Example 2 Exorphin Inhibitor Composition

An exorphin-inhibiting composition was created comprising casomorphinase(having DPP IV activity) and gluteomorphinase, and having the followingcomponents.

Peptidase FPII, having an activity of 25,000 HUT (hemoglobin units oftyrosine; National Enzyme Company, Forsythe, Mo., USA 65653, and fromValley Research, Inc., South Bend, Ind., USA 46624 as Validase®FP) andcomprising casomorphinase (having DPP IV activity). It was produced by acontrolled surface fermentation of Aspergillus oryzae on a wheat brankoji culture, followed by extraction with water and then furtherpurification. The peptidase FPII fraction was a white tan, free flowingpowder with no offensive odor, had a protease activity of pH 7.0 of NLT110,000 units/g, a peptidase activity of pH 5.0 of NLT 6,500 units/g, adrying loss of NMT 10%, a condition loss of NMT 10%, heavy metals of NMT50 PPM, arsenic of NMT 2 PPM, a total plate count of NMT 1,000/g, andwas negative for coliforms. The protease activity was determined by amodified Anson-Hajiwara procedure using a Hammerstein casein substrate,while the peptidase activity was determined using synthetic substrate,H-Glu-Tyr-Glu-OH. The Peptidase FPII composition additionally comprisedother enzyme activities, including an alkaline protease, a neutralprotease, and endoproteases, as well as leucine amino peptidases.Optimum pH was about 6-9 and its stable pH was about 2.8-10.1.

Acid-stable protease ((25 SAPU) Spectrophotometric Acid Protease Units);Bio-Cat, Inc., Troy, Va., USA 22974, and National Enzyme Company,Forsythe, Mo., USA 65653), which is a protease having a higher activityprofile at lower pH levels. This component provided continued andadditional proteolysis in the acid conditions of the stomach. The enzymewas manufactured with wheat bran culture of Aspergillus and wasextracted with water and further purified using ethanol. It was ayellowish powder having an activity of 15,000 units/g wherein one unitof acid protease activity is defined as the quantity of enzyme needed toproduce amino acids equivalent to 100 μmol of tyrosine in 1 ml offiltrate per 60 minutes of 37° C. and pH 3.0.

Protease 20,000 HUT (National Enzyme Company, Forsythe, Mo., USA 65653,and from Valley Research, Inc., South Bend, Ind., USA 46624 asValidase®FP 600). This enzyme was produced by a controlled fermentationof Aspergillus oryzae and contains both endopeptidase and exopeptidaseactivity. Endopeptidases hydrolyze the interior peptide bonds of proteinliberating peptides of varying lengths, and the exopeptidases liberateamino acids by hydrolysis of the peptide bonds at the terminus of thepeptide chain. The enzymes have a broad substrate specificity for, e.g.,gluten, egg yolk, casein, soya, gelatin, hemoglobin and fish. It has anactivity of 500,000 HUT/g, is in a light tan powder and is soluble inwater. (One hemoglobin unit on the tyrosine basis (HUT/g) is thatactivity which produces, in one minute, hydrolysate that has anabsorbency at 275 nm equivalent to that of a solution containing 1 μg/mlof tyrosine in 0.006 N hydrochloric acid.) This component had an optimumpH range of 2.5 to 6.0 30° C.

Lactase, 1,000 ALU (Acid Lactase Units; National Enzyme Company,Forsythe, Mo., USA 65653; Valley Research, Inc., South Bend, Ind., USA46624). Lactase obtained by a fermentation of Aspergillus oryzae andcatalyzed the hydrolysis of lactose beta-D-galactoside linkage,liberating one mole of D-glucose and one mole of D-galactose. Thecomponent has 100,000 F.C.C. LU (F.C.C. lactase unit)/g, is a light tanamorphous dry powder and is free of offensive odor and taste. 1 F.C.C.LU is that amount of enzyme that will liberate one micromole ofo-nitrophenol per minute at pH 4.5 and 37° C. The lactase has aneffective pH range of 3.5-6.5 and optimum pH range of 4.5-5.0.

Papain (sulfite free), 2,500,000 F.C.C. PU (Food Chemical Codex PlantUnits; National Enzyme Company, Forsythe, Mo., USA 65653). Papain has arelatively broad substrate specificity including substrates containing abulky non-polar side chain (such as phenylalanine) at the P2 position ofa P1-2 cleavage site.

L-lysine, 100/mg. L-lysine is an amino acid with alkaline properties. Itis believed to provide a more alkaline microenvironment for the otherenzymes while in the stomach, and thereby tends to increase enzymeactivity.

Example 3 Administration of Exorphin-Inhibiting Composition to HumanAutism Patients

The composition of Example 2 was formulated into number 1-size gelatincapsules containing 360 mg of active ingredients plus a rice bran base.The formulation contained no gluten, casein, soy, corn, sugars, flavors,fragrances, preservatives, salicylates, maltodextrin, artificial colors,or other common allergenic substances. One capsule was taken at thebeginning of each meal. If the capsules could not be swallowed theycould be opened (pulled apart), and the contents added to the firstseveral spoonfuls of food of each meal. The enzyme was not added to allthe food or to portions that would not be eaten immediately.

Capsules were taken at the beginning of all meals and snacks (other thandrinks: water, juice, colas, etc.). Continual snacking was discouragedas it places a continual demand on the production of digestive enzymes.If the individual forgot to take the capsule at the beginning of a meal,it was taken during or up to 15 minutes after the meal, but thedigestive effects were possible lessened in this instance.

The composition was administered to the patients as described above forfour weeks.

Example 4 Results of Administration to Human Patients

The results of the administration in Example 3 indicated that asubstantial number of the participants noted significant improvement oftheir symptoms, as shown in Table 1.

TABLE 1 Symptom 0 1 2 3 4 5 Blank ** Eye Contact 17 14 6 2 1 0 3 21%Socialization 12 15 12 1 1 0 2 33% Attention 16 15 7 2 1 0 3 23% Mood 238 6 2 0 0 4 19% Hyperactivity 27 7 1 2 0 0 6 7% Anxiety 30 5 1 2 0 0 67% Stimming 25 5 1 1 1 0 10 7% Comprehension 11 20 3 3 1 0 6 16% Speech17 13 7 1 2 0 3 23% Sound Sensitivity 27 5 1 0 1 0 9 5% Digestion 19 7 52 1 0 9 19% Sleep 24 7 2 1 0 0 9 7% Perseveration 26 4 0 4 0 0 9 9%Improvement Ratings: 0 = none 1 = possible 2 = moderate 3 = significant4 = great 5 supplied by participant Blank = no comment on survey**percentage of group total reporting 2 to 5 improvement rating.

These results indicated that a “dose response” relationship may bepresent. Such a dose response would reflect that the amount of enzymewas adequate to result in significant changes in some participants, butthat such patients would require a higher dose before symptoms wereaffected. The results also indicated that there may be a certainpercentage of children who do not have problems with gluten or caseinand use of the composition of Example 2 may assist with bowel functionand digestibility of foods but not affect the autism symptoms.

The results also demonstrated that the composition of Example 2 wasgenerally very well tolerated and quite safe. Except for two cases itwas noted that adverse responses were generally transient and did notcontinue throughout the full four weeks. In evaluating the two whoexperienced these responses, there may have been other factors thatcould have contributed to some of the symptoms.

In order to determine if a dose relationship may be present, the dosagewas increased and again administered to participants, as shown in thefollowing Example 5.

Example 5 Administration of Exorphin-Inhibiting Composition to HumanAutism Patients

The composition of Example 2 was formulated (as in Example 3) intoNumber 1-size gelatin capsules containing 360 mg of active ingredientsplus a rice bran base. The capsules were administered to patients asdescribed in Example 3, except that two capsules were administered atthe beginning of meals. After two weeks, the following results werereported:

TABLE 2 Week 1 Week 2 Symptom N₁ = 20 % N₁ = 17 %* Eye Contact 10 50 1155 Socialization 11 55 12 60 Attention 10 50 8 40 Mood 6 30 8 40Hyperactivity 6 30 4 20 Anxiety/Compulsions 6 25 6 30 Stimming 3 15 3 15Speech 11 55 9 45 Sound Sensitivity 2 10 4 20 Digestion 6 30 3 15 Sleep5 25 3 15 Perseveration 4 20 5 25 Individuals not helped 3 15 2 10 atall Helped moderately 9 45 20 50 or significantly *All % are of N¹

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. A composition for reducing the symptoms of autism in a human patient,comprising a physiologically effective amount of a purified casomorphininhibitor that inhibits the opioid bioactivity of casomorphin by actionof a casomorphinase which is a dipeptidyl peptidase, and aphysiologically effective amount of a purified gluteomorphin inhibitorthat inhibits the opioid bioactivity of gluteomorphin by action of agluteomorphinase which is a phenylalaninase, and at least one of thegroup consisting of a physiologically acceptable carrier, adjuvant,excipient, buffer and diluent, wherein the composition decreases theincidence of one or more symptoms of autism selected from the group ofsymptoms consisting of eye contact avoidance, failure to socialize,attention deficit, poor mood, hyperactivity, anxiety, stimming, poorcomprehension, inappropriate speech, abnormal sound sensitivity, poordigestion, disrupted sleep, and perseveration, and wherein thecomposition is suitable for oral administration and the decreasedincidence is measured relative to the incidence in the untreatedindividual.
 2. The composition of claim 1 wherein the dipeptidylpeptidase is a dipeptidyl peptidase IV.
 3. The composition of claim 1wherein the casomorphin inhibitor and the gluteomorphin inhibitor arenot enterically coated.
 4. The composition of claim 1 wherein thecasomorphin inhibitor and the gluteomorphin inhibitor are notenterically coated.
 5. The composition of claim 2 wherein thecasomorphin inhibitor and the gluteomorphin inhibitor are entericallycoated.
 6. The composition of claim 2 wherein the casomorphin inhibitorand the gluteomorphin inhibitor are enterically coated.
 7. Thecomposition of claim 1 wherein at least one of the group consisting of aphysiologically acceptable carrier, adjuvant, excipient, buffer anddiluent comprises a maltodextrin, L-lysine or lactose.
 8. Thecomposition of claim 1 wherein the composition further comprises atleast one non-specific protease.
 9. The composition of claim 8 whereinat least one non-specific protease comprises papain and at least oneother non-specific protease.
 10. The composition of claim 8 wherein thecomposition further comprises at least one lipase, and at least onecarbohydratase.
 11. The composition of claim 10 wherein at least onenon-specific protease comprises papain and at least one othernon-specific protease, and at least one carbohydratase comprises lactaseand at least one other carbohydratase.
 12. A method of reducing thesymptoms of autism in a human patient, comprising orally administeringto the patient a composition comprising a physiologically effectiveamount of a purified casomorphin inhibitor that inhibits the opioidbioactivity of casomorphin by action of a casomorphinase which is adipeptidyl peptidase, and a physiologically effective amount of apurified gluteomorphin inhibitor that inhibits the opioid bioactivity ofgluteomorphin by action of a gluteomorphinase which is aphenylalaninase, and at least one of the group consisting of aphysiologically acceptable carrier, adjuvant, excipient, buffer anddiluent, wherein the composition decreases the incidence of one or moresymptoms of autism selected from the group of symptoms consisting of eyecontact avoidance, failure to socialize, attention deficit, poor mood,hyperactivity, anxiety, stimming, poor comprehension, inappropriatespeech, abnormal sound sensitivity, poor digestion, disrupted sleep, andperseveration, and wherein the composition is suitable for oraladministration and the decreased incidence is measured relative to theincidence in the untreated individual.
 13. The method of claim 12wherein the peptidase comprises a dipeptidase activity of dipeptidylpeptidase IV.
 14. The method of claim 12 wherein the casomorphinase andthe gluteomorphinase are not enterically coated.
 15. The method of claim12 wherein the casomorphinase and the gluteomorphinase are entericallycoated.
 16. The method of claim 13 wherein the casomorphinase and thegluteomorphinase are not enterically coated.
 17. The method of claim 13wherein the casomorphinase and the gluteomorphinase are entericallycoated.
 18. The method of claim 12 wherein at least one of the groupconsisting of a physiologically acceptable carrier, adjuvant, excipient,buffer and diluent enhances the oral administration of the compositionto a human patient.
 19. The method of claim 12 wherein at least one ofthe group consisting of a physiologically acceptable carrier, adjuvant,excipient, buffer and diluent comprises a maltodextrin, L-lysine orlactose.
 20. The method of claim 12 wherein the composition furthercomprises at least one non-specific protease.
 21. The method of claim 20wherein the composition further comprises at least one lipase, and atleast one carbohydratase.
 22. The composition of claim 20 wherein atleast one non-specific protease comprises papain and at least one othernon-specific protease.
 23. The composition of claim 21 wherein at leastone non-specific protease comprises papain and at least one othernon-specific protease, and at least one carbohydratase comprises lactaseand at least one other carbohydratase.
 24. A method of manufacturing amedicament for reducing the symptoms of autism in a human patient,comprising combining physiologically effective amount of a purifiedcasomorphin inhibitor that inhibits the opioid bioactivity ofcasomorphin by action of a casomorphinase which is a dipeptidylpeptidase, and a physiologically effective amount of a purifiedgluteomorphin inhibitor that inhibits the opioid bioactivity ofgluteomorphin by action of a gluteomorphinase which is aphenylalaninase, and at least one of the group consisting of aphysiologically acceptable carrier, adjuvant, excipient, buffer anddiluent, wherein the medicament decreases the incidence of one or moresymptoms of autism selected from the group of symptoms consisting of eyecontact avoidance, failure to socialize, attention deficit, poor mood,hyperactivity, anxiety, stimming, poor comprehension, inappropriatespeech, abnormal sound sensitivity, poor digestion, disrupted sleep, andperseveration, and wherein the composition is suitable for oraladministration and the decreased incidence is measured relative to theincidence in the untreated individual.
 25. A kit comprising a vesselcontaining a composition according to claim 1 and instructions directingthe use of the composition to reduce the symptoms of autism in a humanpatient.
 26. A vessel containing a composition according to claim 1 anda label comprising instructions directing the use of the composition toreduce the symptoms of autism in a human patient.